1. Field of the Invention
This invention relates to an improved process for the production of acrylamide using microorganisms.
2. Description of the Prior Art
It has long been known that microorganisms having nitrilasic activity are effective in hydrolyzing acrylonitrile to yield acrylamide. As such microorganisms, those belonging to the genera Bacillus, Bacteridium in the sense of Prevot, Micrococcus, Brevibacterium in the sense of Bergey, etc., are known (see, for example, U.S. Pat. No. 4,001,081). It has also been discovered by the inventor that those microorganisms belonging to the genera Corynebacterium and Nocardia are also useful for the hydrolysis of acrylonitrile, as described in Japanese Patent Application (OPI) No. 129190/1979 (the term "OPI" as used herein refers to a "published unexamined Japanese patent application").
The production of acrylamide from acrylonitrile by use of such microorganisms is carried out by bringing the microorganism, either as is or after being fixed on a polymeric gel, into contact with acrylonitrile in an aqueous medium (e.g., water, a physiological saline solution, a phosphate buffer solution, etc.). Recently, microorganic reactions using a batchwise or continuous method using granulated fixed cells have been in widespread use for the purposes of the prevention of elution of impurities from the cells, the separation of the cells from a reaction solution, repeated utilization of the cells, an increase in the stability of enzymes, etc. In the production of acrylamide by the use of microorganisms, such methods using granulated fixed cells are advantageous from an economic viewpoint, and the inventors have proposed the production of acrylamide by a continuous column reaction using cells which are fixed by entrapping them with a gel of polyacrylamide, as described in Japanese Patent Application (OPI) No. 143593/1979.
However, the use as an aqueous medium of the above-described physiological saline solution, phosphate buffer solution, etc., is not preferred, in that the acrylamide aqueous solution formed contains large amounts of sodium chloride, phosphoric acid salts, etc., leading to the formation of low quality acrylamide. In particular, when acrylamide of a solution containing phosphoric acid salts is polymerized to produce acrylamide based polymers having high degrees of polymerization, the acrylamide based polymers formed are undesirably liable to become insoluble in water. Therefore, post-treatments such as an ion exchange treatment, etc., become essential for the removal of the phosphoric acid salts prior to polymerization. This leads to the loss of the advantage that a high quality acrylamide aqueous solution can be obtained without providing any special purification step, which is a feature of the method of producing acrylamide by a fixed cells method. Thus, the advantage of the fixed cells method as an inexpensive method for the production of acrylamide is lost.
On the other hand, if the physiological saline solution, phosphate buffer solutions, or the like is not used as the aqueous medium, the fixed cells swell and the enzymatic activity of the cells is rapidly lost. Furthermore, in a continuous hydration reaction of acrylonitrile by use of a column charged with microorganic cells which are fixed by the gel entrapping method, the fixed cells in the column swell in a short time after the start of the reaction, as a result of which efficient operation of the process becomes impossible.
Although the reason why the fixed cells swell during the hydration reaction is not clear, it is believed to be due to the difference in osmotic pressure between the outside and inside of the fixed cells, which results from the difference in concentrations of acrylonitrile and acrylamide between the outside and inside of the cell. This difference is caused to occur when acrylonitrile enters the inside of the fixed cells and is converted or hydrated into acrylamide, and the thus-formed acrylamide goes out of the fixed cells. Furthermore, it is also believed to be due to the facts that the enzyme is liable to leak out of the cell due to swelling and that the stable conformation in normal cells in which the enzyme is not swollen cannot be maintained. Therefore, it is believed that when the reaction is carried out in an isotonic medium comprising a physiological saline solution, a phosphate buffer solution, etc., no great difference in osmotic pressure between the outside and inside of the fixed cells is created, and thus the swelling of the cells of the fixed cells can be prevented.